Cytokine-binding domain

ABSTRACT

The present invention provides a cytokine-binding domain or portion thereof which binds to at least one cytokine and is capable of transducing a cytokine signal through a single cytokine receptor, said domain comprising a portion of the B′-C′ loop of domain 4 of a β c  chain or analogous structure of a cytokine receptor. In another aspect of the invention there is provided a method of identifying a compound having cytokine agonist or antagonist activity, said method including subjecting a potential cytokine agonist and/or cytokine antagonist compound to a cytokine binding domain or portion thereof wherein said domain binds to at least one cytokine and is capable of transducing a cytokine signal through a single cytokine receptor, said domain comprising a portion of the B′-C′ loop of domain 4 of a β c  chain or analogous structure of the cytokine receptor; and determining the presence of an agonist or antagonist response of the compound on the activity of a cytokine. In a preferred aspect there is provided a method fo identifying a GM-CSF, IL-3 and IL-5 agonist or antagonist, said method including: subjecting a potential agonist or antagonist to a GM-CSF, IL-3 and IL-5 binding domain or portion thereof wherein said domain binds to at least one of the cytokines and is capable of transducing a cytokine signal through a single cytokine receptor, said domain comprising a portion of the B′-C′ loop of domain 4 of a β c  chain or analogous structure of the cytokine receptor; and determining the presence of a agonist or antagonist response from the compound on the activity of GM-CSF, IL-3 and IL-5/

[0001] The present invention relates to a cytokine binding domain, andto a cytokine-binding antagonist and/or a cytokine-binding agonist. Theinvention further relates to methods of identifying such compounds anduses of such compounds in therapy, prevention and diagnosis.

BACKGROUND OF THE INVENTION

[0002] Heterodimeric cytokine receptors comprise two (or three) subunitswhich subserve distinct and specialised functions. These include a majorligand-binding subunit (the α subunit) and a signalling subunit (the βor γ subunit). Importantly, the latter is able to recognise severalcytokines complexed to the appropriate α chain and transduce theirsignals. This is exemplified by the common β chain (β_(c)) of the humangranulocyte-macrophage colony-stimulating factor GM-CSF, interleukin-3(IL-3) and IL-5 receptors, the common IL-2 receptor β chain (shared bythe IL-2, IL-4, IL-7, IL-9 and IL-15 receptors) and gp130 (shared by theIL-6, IL-11, LIF, ciliary neutrophic factor, oncostatin M andcardiotrophin receptors). Significantly, IL-5, IL-3 and GM-CSF, the onlythree cytokines known to stimulate eosinophil production, can be foundconcomitantly elevated in lungs affected by allergic inflammation.

[0003] The simultaneous antagonism of all three GM-CSF, IL-3 and IL-5may be desirable or indeed necessary for stimulating eosinophils. Forexample, eosinophils which are believed to be the major cell typeinvolved in allergy can be maintained in numbers and be stimulated byeither IL-3, GM-CSF or IL-5 (Lopez et al, 1989). Antagonism of all threecytokines may thus be necessary to inhibit the actions of eosinophilsand basophils. Similarly, basophils which are also believed to play aneffector role in allergy can be stimulated by either IL-3, GM-CSF orIL-5 (Lopez et al, 1990). Antagonism of GM-CSF, IL-3 and IL-5 may beaccomplished by the concomitant administration of specific antagonistsfor each different cytokine. Though feasible, this approach has thedisadvantage of having to administer up to three different proteinswhich is not only inconvenient but which also increases the risk ofimmunogenicity and other side-effects.

[0004] Because all three of these cytokines act through a commonreceptor subunit (β_(c)) it may be possible to simultaneously inhibitthe action of GM-CSF, IL-3 and IL-5 with a single compound via the(β_(c)) subunit.

[0005] Thus, an antagonist directed against the β_(c) chain maysimultaneously inhibit the function of all three cytokines and may provea useful therapeutic.

[0006] One of the major problems in seeking structural data of thebinding site of a communal subunit complexed to cytokines is that,unlike homodimeric receptors or isolated α chains of heterodimericreceptors which can directly bind to cytokines, communal subunits cannotbind to cytokines by themselves. To overcome this problem applicantshave developed a monoclonal antibody (Mab) against a region which isimportant for cytokine high affinity binding within domain 4 of theGM-CSF/IL-3/IL-5 common beta chain (D4β_(c)) receptor. This Mab, termedBION-1, inhibited the high affinity binding of GM-CSF, IL-3 and IL-5 tohuman eosinophils, and inhibited their in vitro production andfunctional activation. BION-1 thus represents the first commonantagonist of the GM-CSF, IL-3 and IL-5 receptors and a unique tool withwhich to explore the cytokine-binding site in the common beta chain.

[0007] The molecular basis for the affinity conversion of β_(c) to eachligand is not fully understood as the ligand-receptor complex had notyet been crystallised and this has prevented the structural definitionof their ligand-binding sites. Applicants have now crystallised anddetermined the structure of the D4β_(c) domain of the GM-CSF/IL-3/IL-5receptor bound to an antagonist in the form of BION-1.

SUMMARY OF THE INVENTION

[0008] The present invention provides a cytokine-binding domain orportion thereof which binds to at least one cytokine and is capable oftransducing a cytokine signal through a single cytokine receptor, saiddomain comprising a portion of the B′-C′ loop of domain 4 of a β_(c)chain (D4β_(c)) or analogous structure of a cytokine receptor.

[0009] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0010] In another embodiment, the domain further includes a Tyrosineresidue capable of interaction with an α chain subunit or with Domain 3of the β_(c) chain subunit to allow high affinity binding of thecytokine. In a further preferred embodiment, the tyrosine is Tyr 421 orequivalent residue. The Tyr 421 or equivalent residue improves β_(c)intramolecular interactions and/or the receptor subunit-receptor subunitinteraction or oligomerisation.

[0011] Preferably the binding domain is capable of recognising at leasttwo cytokines that have complexed to an α chain. Preferably thesecytokines are selected from the group including, but are not limited to,IL-3, IL-5 and GM-CSF or from IL-4 and IL-13. The cytokines particularlyIL-3, IL-5 and GM-CSF may bind via the common β_(c) chain of thecytokine receptor having firstly been bound by the α chain and forming acytokine: α chain complex.

[0012] In another aspect of the invention there is provided a method ofidentifying a compound having cytokine agonist or antagonist activitysaid method including:

[0013] subjecting a potential cytokine agonist and/or cytokineantagonist compound to a cytokine binding domain or portion thereofwherein said domain binds to at least one cytokine and is capable oftransducing a cytokine signal through a single cytokine receptor, saiddomain comprising a portion of the B′-C′ loop of domain 4 of a β_(c)chain or analogous structure of the cytokine receptor; and

[0014] determining the presence of an agonist or antagonist responsefrom the compound on the activity of a cytokine.

[0015] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0016] The cytokine binding domain may be a domain of a receptor commonto a number of cytokines. Preferably the domain is common to GM-CSF,IL-3 and IL-5 or is a common signalling structure common to IL-4 andIL-13.

[0017] In a preferred aspect there is provided a method of identifying aGM-CSF, IL-3 and IL-5 agonist or antagonist said method including:

[0018] subjecting a potential agonist or antagonist to a GM-CSF, IL-3and IL-5 binding domain or portion thereof wherein said domain binds toat least one of the cytokines and is capable of transducing a cytokinesignal through a single cytokine receptor, said domain comprising aportion of the B′-C′ loop of domain 4 of a β_(c) chain or analogousstructure of the cytokine receptor; and

[0019] determining the presence of an agonist or antagonist responsefrom the compound on the activity of GM-CSF, IL-3 and IL-5.

[0020] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0021] In another preferred aspect of the invention there is provided amethod of identifying a compound having a cytokine antagonist activity,said method including:

[0022] subjecting a potential cytokine antagonist to a cytokine bindingdomain or portion thereof wherein said domain or portion thereof bindsto at least one cytokine and is capable of transducing a cytokine signalthrough a single cytokine receptor, said domain comprising a portion ofthe B′-C′ loop of domain 4 of a β_(c) chain or analogous structure ofthe cytokine receptor; and

[0023] identifying a compound that has bound to the cytokine-bindingdomain wherein said compound has an antagonist response on the activityof the cytokine.

[0024] More preferably, the domain comprises a portion of the B′-C′loopof domain 4 and groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0025] In a preferred embodiment, there is provided a method foridentifying an antagonist of GM-CSF, IL-3 and IL-5, said methodincluding:

[0026] subjecting a potential cytokine antagonist to a cytokine bindingdomain or portion thereof wherein said domain or portion thereof bindsto at least one of the cytokines and is capable of transducing acytokine signal through a single cytokine receptor, said domaincomprising a portion of the B′-C′ loop of domain 4 of a β_(c) chain oranalogous structure of the cytokine receptor; and

[0027] identifying a compound that has bound to the cytokine-bindingdomain wherein said compound has an antagonist response on the activityof the cytokine.

[0028] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0029] In another aspect of the invention there is provided a method ofpreventing or treating a cytokine—related condition, said methodincluding administering to a subject an effective amount of a compound,agonist or antagonist identified by the methods as described above.

DESCRIPTION OF THE FIGURES

[0030]FIG. 1 illustrates the structure of D4β_(c). (A) Structure of theFab/receptor β_(c) domain 4 (D4β_(c)) complex shown in ribbonrepresentation. The MoAb heavy chain is shown in dark grey, the lightchain and the receptor in light grey. The major structural features ofD4β_(c) are labelled and the locations of key residues are denoted bystick representation. These pictures were produced using Molscript(Kraulis, 1991) and Raster3D (Merrift and Murphy, 1994). (B) Structureas for (A) but reoriented 90° about the vertical axis. (C) Surfacerepresentation of the receptor using the program GRASP³⁵. The darksurface indicates the location of hydrophobic/aromatic patch, H₁. Themolecule is tilted approximately 20° counterclockwise relative to (A).(D) View of hydrophobic/aromatic patch, H2 prepared as for (C). Themolecule is tilted approximately 20° clockwise and rotated approximately60° clockwise from above about a vertical axis relative to (B).

[0031]FIG. 2 illustrates a view of the Trp/Arg “ladder”. The structureof the D4β_(c) shown in ribbon representation with the side-chains ofthe Trp/Arg stack shown as ball-and-stick. The molecular graphics wereproduced using Molscript (Kraulis, 1991) and Raster3D (Merritt, andMurphy, 1994).

[0032]FIG. 3 illustrates a surface representation of the hexamericGM-CSF receptor complex model, looking side-on (A) and from above (B). αchain is in red, β chain in yellow, and each GM-CSF monomer in magentaand cyan.

[0033]FIG. 4 illustrates the interactions and interface between theantibody (BION-1) and D4β_(c).

[0034]FIG. 5 illustrates a stereoview 2F_(obs)-F_(calc) electron densitymaps showing key regions of the receptor. The maps were calculated fromthe final model and contoured at 1c. (A) The B′-C′ loop. (B) Theextended WSXWS box.

[0035]FIG. 6 illustrates the differential effects of mutating the B-Cloop and/or Tyr⁴²¹ of the F-G loop in receptor activation. (A) Scatchardtransformation of binding isotherms for ¹²⁵I-GM-CSF and ¹²⁵I-IL-3binding to cells transfected with wild type β_(c) (E) or ³⁶⁵AAAA³⁶⁸mutant β_(c) ( ). (B) Western blot of wild type and mutant β_(c) afterstimulation with various concentrations of IL-3. The blot was probed forphosphotyrosine (upper panel) and β_(c) (lower panel). The double bandsin each lane of the gels represent glycosylation variants of β_(c)(Woodcock, J. M. et al (1997) Blood 90: 3005).

[0036]FIG. 7 illustrates a comparison of D4β_(c) with themembrane-proximal domain of GHR. D4β_(c) and domain two of the subunitof the GHR that interacts with the helix A/helix C face of GH werealigned structurally via their core residues and are shown as surfacerepresentations using the program InsightII (MSI). Thehydrophobic/aromatic patch, H2, of D4β_(c) and the location of the siteof GHR that interacts with the opposing receptor molecule are indicatedby darkened surfaces and are ringed and labelled “Receptor interfaceresidues. The dark surfaces of D4β_(c) indicating the region known tointeract with GH are ringed and labelled “Ligand-binding residues”.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides a cytokine-binding domain orportion thereof which binds to at least one cytokine and is capable oftransducing a cytokine signal through a single cytokine receptor, saiddomain comprising a portion of the B′-C′ loop of domain 4 of a β_(c)chain or analogous structure of a cytokine receptor.

[0038] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure of acytokine receptor.

[0039] In another embodiment, the domain further includes a Tyrosineresidue capable of interaction with an α chain subunit or with Domain 3of the β_(c) chain subunit to allow high affinity binding of thecytokine. In a further preferred embodiment, the tyrosine is Tyr 421 orequivalent residue. The Tyr 421 or equivalent residue improves β_(c)intramolecular interactions and/or the receptor subunit-receptor subunitinteraction or oligomerisation.

[0040] The term “chain” and “chain subunit” may be used interchangeablythroughout the specification. For instance, the “α chain” is the same asthe “α chain subunit.”

[0041] Throughout the description and claims of the specification theword “comprise” and variations of the word, such as “comprising” and“comprises” is not intended to exclude other additives, components,integers or steps.

[0042] The term “equivalent residue” used herein means an amino acidresidue which can perform a similar function.

[0043] Preferably the binding domain is capable of recognising andbinding to at least two cytokines that have complexed to an appropriateα chain. Preferably these cytokines are selected from the groupincluding, but are not limited to, IL-3, IL-5 and GM-CSF or to IL-4 andIL-13 The three cytokines IL-3, IL-5 and GM-CSF may be bound via thecommon β_(c) chain of the cytokine receptor having firstly been bound bythe α chain and forming a cytokine: α chain complex. Similarly, IL-4 andIL-13 may share a common signalling unit similar to the β_(c) on thecytokine receptor.

[0044] The common β_(c) chain may derive from any one of the following,including GM-CSF, IL-3 and IL-5 receptors. Common signalling subunitshaving similar structures to β_(c) may be derived from the common IL-2receptor γ chain (shared by the IL-2, IL-4, IL-7, IL-9 and IL-15receptors) and gp130 (shared by the IL-6, IL-11, LIF, ciliary neutrophicfactor, oncostatin M and cardiotrophin receptors) or from any one of thecytokine superfamily receptors but not limited to the group comprisingLIFR, gp130, IL-2Rβ, IL-4R/IL-13R, IL-2Rγ, IL-3Rα, EPOR, TPOR and OBR orbe selected from a related (class 1) cytokine receptor structureselected from the group including but not limited to growth hormonereceptor (GHR), prolactin receptor (PRLR), erythropoietin receptor(EPOR), G-CSF receptor (G-CSFR) and gp130. The pairwise sequenceidentities between D4β_(βc) and these receptors, after structure-basedalignment, range from 12% (G-CSF) to 27% (gp130). There are only sevenresidues (Pro 343, Trp 358, Leu 402, Tyr 408, Arg 413, Gly 423, Ser 426)that are strictly conserved across the receptors, all of which appear toplay structural roles. A structural superposition indicates that D4βc ismost closely related to PRLR (r.m.s. deviation of 1.4 Å on 86 Ca atoms,20% sequence identity) followed by GHR (r.m.s. deviation of 1.5 Å on 81Ca atoms, 23% sequence identity).

[0045] Preferably, the common β_(c) chain is derived from IL-5, IL-3 orGM-CSF receptor. These cytokines IL-3, IL-5 and GM-CSM are known tostimulate eosinophil production and can be found concomitantly elevatedin lungs affected by allergic inflammation. Their simultaneous elevationmay increase eosinophil numbers, contribute to the overall degree ofeosinophil activation, be responsible for the different phases ofeosinophil infiltration and determine a localised versus a generalisedeosinophil-mediated inflammation. This may be particularly important inthe pathology of certain disease such as asthma where the eosinophilplays an effector role.

[0046] The present invention describes the binding domain of the β_(c).However this is illustrative only and is not to be considered alimitation on the invention described.

[0047] Applicants expressed the fourth (activation) domain of β_(c)(D4β_(c)) in E. coli and purified it to homogeneity by ion exchange andreverse-phase high performance liquid chromatography. BION-1 MoAb wasdigested with papain to generate Fab fragments which were purified bychromatography on protein A sepharose. Titration of D^(4β) _(c) and theBION-1 Fab produced a stoichiometric 1:1 complex that subsequentlyformed crystals. These crystals diffracted well enough to allow a fullcrystallographic structure determination to proceed.

[0048] The common β_(c) chain of domain 4 (D4β_(c)) has been found bythe Applicants to have a compact globular shape with overall dimensionsof 45 Å×25 Å×20 Å (FIG. 1A). The N- and C-termini represent the sites ofattachment for the remainder of the extracellular region and themembrane-spanning domain, respectively. The molecule adopts the topologyof a fibronectin type III module with two anti-parallel beta-sheets (42%sheet) packing against each other via a multitude of hydrophobicinteractions, including two clusters of aromatic residues (Trp 434, Tyr354 and Tyr 376; Trp 358, Phe 372 and His 370). Sheet A consists ofthree beta-strands (A′(residues 344 to 350), B′(residues 353 to 359) andE′(residues 396 to 398)) and sheet B consists of four strands(C′(residues 369 to 378), D′(residues 389 to 392), F′ and F″(residues406 to 417) and G′(residues 432 to 436)) with the longest strand, C′,almost spanning the length of the molecule. The amino acid sequencemotif, WSXWS (where X is any residue), a characteristic feature of manycytokine receptors, is located between the F strand, preferably the F″and the G strand, preferably the G′ strands and adopts a double β-bulgestructure (FIG. 1A). Arginine residues from strand F′ interdigitatebetween the tryptophan residues of the motif to form a ladder ofalternating basic and aromatic residues. The ladder is extended in eachdirection by additional aromatic and basic residues: Tyr 421-Arg 415-Trp425-Arg 413-Trp 428-Arg 411-Trp 383-Arg 377-Trp 409-Arg 407. There is a“side-step” in the ladder at Arg 377-Trp 409. This ten-rung ladder,measuring 29 to 35 Å long, preferably 29 Å long with rungs of about 5 Åwide, represents the only significant electropositive patch on thesurface of the molecule.

[0049] The crystallographic analysis is provided below in Table 1. TABLE1 Crystallographic analysis Data collection Temperature of collection100 Multiplicity 2.8 (K) l/σ_(I) 11.4 Resolution limit (Å) 2.8 No. ofdata >2σ_(I) (%) 66.2 Observations 58,732 R_(merge) ^(a) (%) 9.8 Uniquereflections 21,211 Completeness (%) 88.1 Refinement statisticsResolution range ∞-2.8 r.m.s. deviations from (Å) ideality R_(factor)^(b) (%) 22.8 bond lengths (Å) 0.010 R_(free) ^(b) (%) 28.8 bond angles(°) 1.55 impropers (°) 0.95 dihedrals (°) 27.1 Atoms in model protein(non-hydrogen) 4,146 water 124 carbohydrate 14 Residues in most favored^(c) regions of Ramachandran plot (%) 80 Residues in additionallyallowed ^(c) regions of Ramachandran plot (%) 19

[0050] Besides the ladder, there are three other significant surfacefeatures worth noting. There are two large hydrophobic patches on thesurface. The first, H1, is a dense strip of hydrophobic residues locatedat one edge of the β-sandwich defined by the D′ and E′ strands andmeasures 27 Å long and 6 Å in width (FIG. 1D). The second, H2, locatedon the opposite face to the first, forms part of a lip at the end of apronounced groove on the surface of the molecule (FIG. 1C). The H2 patchis made up of residues Ile 338, Ala 341, Met 361, Tyr 365, andpreferably including Met 340 and Pro 342 and the aliphatic moiety of Lys362 and preferably including Ile 368 and Tyr 421. The groove is locatedat the N-terminal end of the molecule where one wall is formed by theB′-C′ loop and part of the F″-G loop and the other wall by theN-terminus (residues 338 to 342) (FIG. 1C).

[0051] The B′-C′ loop of domain 4 or the common β_(c) chain (D4β_(c)) ofthe cytokine receptor or part thereof is involved in the cytokinebinding. The B-C and preferably the F-G loops protrude significantlyfrom the body of the protein and are implicated in cytokine binding(FIGS. 1A,B). It adopts a regular structure in D4β_(c) having residues365 to 368 forming a type Iβ-turn (FIG. 1). Preferably the portion of athe B′-C′ loop of the domain includes Tyr 365, Ile 368 and His 367.GCSFR, GHR, and PRLR have an aromatic residue equivalent to Tyr 365,whereas there is no corresponding residue to His 367.

[0052] In the interaction of a cytokine with the B′-C′ loop it isfurther preferable that the residues Tyr 365, His 367 and Ile 368 areinvolved in the interaction of the cytokine and the receptor. Ideally,the Tyr 365, His 367 and Ile 368 form a cytokine binding triad thatconverges to form a pivot point to which all three cytokines (GM-CSF,IL-3 and IL-5) may bind via essential glutamate residues (Glu 21 ofGM-CSF, Glu 22 of IL-3 and Glu13 of IL-5).

[0053] The F′-G′ loop adopts a type IV β turn at its tip in D4β_(c) andthe most significant features in this region are Arg 418 and Tyr 421,each of which projects out of solution (FIG. 1A). Accordingly, when thecytokine interacts with the β_(c) chain, the Tyr 421 may interact withDomain 3 of β_(c) and/or the α-chain to enhance receptor-receptorinteraction or oligermerisation.

[0054] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4.

[0055] In another embodiment, the domain further includes a Tyrosineresidue capable of interaction with an α chain subunit or with Domain 3of the β_(c) chain subunit to allow high affinity binding of thecytokine. In a further preferred embodiment, the tyrosine is Tyr 421 orequivalent residue. The Tyr 421 or equivalent residue improves β_(c)intramolecular interactions and/or the receptor subunit-receptor subunitinteraction or oligomerisation.

[0056] In a further preferred embodiment the binding domain or portionthereof capable of binding the cytokine may be defined by an areabordered by any of the following residues Lys 362, Tyr 365, His 367, lie368, Arg 418, Gly 420, Asn 422, Thr 416, Ile 338, Gln 339, Met 340 andMet 361 or similar residues of common signalling units in otherreceptors. A majority of these residues are in the B′-C′ loop and henceconstitute a portion of the B′-C′ loop capable of transducing a cytokinesignal. The binding domain may be described as a “groove” comprising aconcave surface formed largely, but not exclusively by hydrophobicresidues, preferably of those listed above.

[0057] The hydrophobic surface patches, H1 and H2, of D4β_(c) (FIGS. 1C,1D) have corresponding features in most of the other receptors. With theexception of gp130, all the receptors possess significant hydrophobicpatches equivalent to the location of H1 (centred about the D′-E′ strandconnection), although the degree and extent of hydrophobicity variesgreatly. The equivalent region to H2 is conserved in all but gp130. Byanalogy with the other receptors, the H2 patch of D4β_(c) might interactwith the A-B loop from domain 3 of the intact receptor.

[0058] The interactions between D4β_(βc) and an antagonist such asBION-1 or a cytokine are summarized in Table 2 below. TABLE 2Interactions between D4β_(c) and BION-1 or cytokines Required forRequired for Residue residue vdw buried Polar binding affinity- identitytype contact¹ area (Å²)² interactions³ BION-1⁴ conversion⁵ D4β_(c) β_(c)361 Met No 0 No No β_(c) 362 Lys Yes 45 Nζ → L94:Oδ No No β_(c) 363 MetYes 121 Sδ → H57:N ? No β_(c) 364 Arg Yes 33 Nη → H33:Oη ? No O → H33:Oηβ_(c) 365 Tyr Yes 87 Oη → L94:Oδ No Yes β_(c) 366 Glu Yes 165 Oε →H35:Nζ Yes No Oε → H33:N β_(c) 367 His Yes 99 Nε → L91:O No Yes β_(c)368 Ile No 16 No Yes β_(c) 369 Asp No 0 No No β_(c) 370 His No 0 No Noβ_(c) 395 His No 26 N.D.⁶ N.D. β_(c) 416 Thr Yes 29 Oγ → L28:Oη N.D.N.D. β_(c) 418 Arg Yes 101 Nη → H97:O Yes No β_(c) 419 Thr No 15 No Noβ_(c) 420 Gly No 0 No No β_(c) 421 Tyr Yes 45 No Yes β_(c) 422 Asn No 0No No BION-1 light chain L  28 Tyr Yes 162 Oη → β_(c)416:Oγ L  29 Gly No21 L  30 Asp No 18 L  32 Phe Yes 39 L  91 Asn Yes 17 O → β_(c)367:Nε L 92 Asn No 14 L  93 Glu No 13 L  94 Asp Yes 48 Oδ → β_(c)362:Nζ Oδ →β_(c)365:Oη L  96 Trp Yes 31 BION-1 heavy chain H  32 Tyr Yes 6 H  33Tyr Yes 100 Oη → β_(c)364:Oη Oη → β_(c)364:O N → β_(c)366:Oε H  35 LysNo 0 Nζ → β_(c)366:Oε H  51A Asn Yes 21 H  53 Asn No 37 H  55 Gly Yes 9H  57 Thr Yes 18 Oγ → β_(c)363:Sδ H  58 Leu No 52 H  96 Asp Yes 6 H  96AGly Yes 51 H  97 Ile Yes 13 O → β_(c)418:Nη H 100A Gly Yes 16

[0059] In another aspect of the invention there is provided a method ofidentifying a compound having cytokine agonist or antagonist activitysaid method including

[0060] subjecting a potential cytokine agonist and/or cytokineantagonist compound to a cytokine binding domain or portion thereofwherein said domain binds to at least one cytokine and is capable oftransducing a cytokine signal through a single cytokine receptor, saiddomain comprising a portion of the B′-C′ loop of domain 4 of a β_(c)chain or analogous structure of the cytokine receptor; and

[0061] determining the presence of an agonist or antagonist response ofthe compound on the activity of a cytokine.

[0062] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0063] In another embodiment, the domain further includes a Tyrosineresidue capable of interaction with an α chain subunit or with Domain 3of the β_(c) chain subunit to allow high affinity binding of thecytokine. In a further preferred embodiment, the tyrosine is Tyr 421 orequivalent residue. The Tyr 421 or equivalent residue improves β_(c)intramolecular interactions and/or the receptor subunit-receptor subunitinteraction or oligomerisation.

[0064] The common β_(c) chain may be common to IL-3, IL-5 or GM-CSF orit may be similar structure common to IL-4 and IL-13.

[0065] The agonist or antagonist response may be measured by the abilityof the compound to bind and activate or inhibit a cytokine response orit may be measured by an inability to bind to a mutant cytokine bindingdomain. This can be measured by cellular activities associated with anyof the cytokines. For instance, with the cytokines IL-5, GM-CSF and IL-3stimulation of eosinophil adherence, priming for degranulation andcytotoxicity, and propagation of viability may be measured in thepresence or absence of the agonists or antagonists binding to thecytokine binding domain or a portion thereof as hereinbefore described.

[0066] In a preferred aspect there is provided a method of identifying aGM-CSF, IL-3 and IL-5 agonist or antagonist said method including:

[0067] subjecting a potential agonist or antagonist to a GM-CSF, IL-3and IL-5 binding domain or portion thereof wherein said domain binds toat least one of the cytokines and is capable of transducing a cytokinesignal through a single cytokine receptor, said domain comprising aportion of the B′-C′ loop of domain 4 of a β_(c) chain or analogousstructure of the cytokine receptor; and

[0068] determining the presence of an agonist or antagonist responsefrom the compound on the activity of GM-CSF, IL-3 and IL-5.

[0069] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0070] In another preferred aspect of the invention there is provided amethod of identifying a compound having a cytokine antagonist activity,said method including:

[0071] subjecting a potential cytokine antagonist to a cytokine bindingdomain or portion thereof wherein said domain or portion thereof bindsto at least one cytokine and is capable of transducing a cytokine signalthrough a single cytokine receptor, said domain comprising a portion ofthe B′-C′ loop of domain 4 of a β_(c) chain or analogous structure ofthe cytokine receptor; and

[0072] identifying a compound that has bound to the cytokine-bindingdomain wherein said compound has an antagonist response on the activityof the cytokine.

[0073] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or an analogous structure.

[0074] In another embodiment, the domain further includes a Tyrosineresidue capable of interaction with an α chain subunit or with Domain 3of the β_(c) chain subunit to allow high affinity binding of thecytokine. In a further preferred embodiment, the tyrosine is Tyr 421 orequivalent residue. The Tyr 421 or equivalent residue improves β_(c)intramolecular interactions and/or the receptor subunit-receptor subunitinteraction or oligomerisation.

[0075] In a preferred embodiment, there is provided a method foridentifying an antagonist of GM-CSF, IL-3 and IL-5, said methodincluding:

[0076] subjecting a potential cytokine antagonist to a cytokine bindingdomain or portion thereof wherein said domain or portion thereof bindsto at least one of the cytokines and is capable of transducing acytokine signal through a single cytokine receptor, said domaincomprising a portion of the B′-C′ loop of domain 4 of a β_(c) chain oranalogous structure of the cytokine receptor; and

[0077] identifying a compound that has bound to the cytokine-bindingdomain wherein said compound has an antagonist response on the activityof the cytokine.

[0078] More preferably, the domain comprises a portion of the B′-C′ loopof domain 4 and a groove which is defined by the B′-C′, F′-G′ loops andthe N-terminal section of domain 4 or analogous structure.

[0079] In another embodiment, the domain further includes a Tyrosineresidue capable of interaction with an α chain subunit or with Domain 3of the β_(c) chain subunit to allow high affinity binding of thecytokine. In a further preferred embodiment, the tyrosine is Tyr 421 orequivalent residue. The Tyr 421 or equivalent residue improves β_(c)intramolecular interactions and/or the receptor subunit-receptor subunitinteraction or oligomerisation.

[0080] The antagonist preferably inhibits the binding of cytokinespreferably IL-3, IL-5 and GM-CSF to the β_(c) or IL-4 and IL-13 to acommon signalling unit similar to β_(c) via a portion of the B′-C′ loopor similar structure. Preferably that portion is identified by the“groove” or part thereof as described above. The cytokines may bind thecommon β_(c) chain or common signalling unit and more particularly to aportion of the B′-C′ loop.

[0081] In a further preferred embodiment, the antagonist may attach,target or block Tyr 421 or equivalent residue, as well as the aminoacids or groups of amino acids that interact with Tyr 421 or equivalentresidue in either the Domain 3 of the β_(c) subunit or the α chainsubunit.

[0082] The methods identified above will allow the identification anddesign of agonists and antagonist of cytokines that can act through theportion of the β_(c) or other common signalling units and/or through theTyr 421 or equivalent residue and/or Domain 3 of the β_(c) chain subunitand/or the α chain subunit of a cytokine receptor or an analogous commonchain in other receptors. Preferably the subunit is common to GM-CSF,IL-3 and IL-5 such as β_(c) or is an analogous chain common in otherreceptors such as in IL-4 and IL-3.

[0083] A crystalline form of the cytokine binding domain is alsoprovided in the present invention and will allow for structure baseddesign of drugs or targeted selection by phage display. Potentialagonists and antagonists may be identified by screening for “groovebinders” (compounds that may bind in the groove). These may bedetermined by considering wild type versus mutant domain 4 molecules.Mutants may be generated using mutations to alanines in the floor of thegroove. Mutations may be directed to any of the residues selected fromthe group including Gin 340, lie 338 and Met 361 to make the mutants.

[0084] Further uses of the structure of domain 4 of the common β_(c)allows for affinity maturation using designed mutations such as those inthe floor of the groove and including mutations at Gln 340, Ile 338and/or Met 361. Because applicants have deduced the structure of domain4, antigen structures are further understood by the present descriptionand development of BION-1 mimetics either peptide or non-peptide isincluded in the scope of this application.

[0085] In another aspect there is provided a compound, agonist orantagonist identified by the methods described above. The compoundagonist or antagonist may be an antibody or fragment thereof directed tothe cytokine binding domain or more preferably the β_(c) chain subunitof the cytokine receptor domain or an analogous common chain in otherreceptors. Even more preferably, the antibody or fragment thereof may bedirected to a portion of the B′-C′ loop of Domain 4 of a β_(c) chainsubunit or analogous structure of a cytokine receptor or the antibodymay be directed to the Tyr 421 or equivalent residue or amino acids orgroups of amino acids that interact with Tyr 421 or equivalent residuein either the Domain 3 of the β_(c) chain subunit or the α chainsubunit. The antibody may be monoclonal or polyclonal or an activeportion thereof.

[0086] Methods of making such antibodies will be familiar to thoseskilled in the art and will be understood to further include the stepsof inoculating an animal with a peptide molecule having the cytokinebinding domain or a portion thereof as described above, fusing antibodyproducing cells with a myeloma cell line and screening for a cell linethat produces an antibody reactive with the cytokine-binding domain orportion thereof, and harvesting antibodies from the cell line, testingfor inhibition of high affinity binding and testing for inhibition orexcitation of function. This may further include making small fragmentsof antibodies produced by the said cell line capable of binding thecytokine binding domain or portion thereof. The cell line mayconveniently be a mouse cell line and the method may include the furtherstep of “humanising” the said antibody fragments by replacing mousesequences with human sequences in the non-binding regions. Humanisingmay be conducted by any methods known to the skilled addressee.

[0087] The antibody fragment may be a larger portion such as Fabfragments or much smaller fragments of the variable region. Thesefragments may be used as separate molecules or alternatively may formpart of a recombinant molecule which is then used for therapeuticpurposes. Thus for example the monoclonal antibody may be “humanised” byrecombining nucleic acid encoding the variable region of the monoclonalantibody with nucleic acid encoding non-variable regions of human originin an appropriate expression vector.

[0088] The agonist and antagonist compounds of the present invention arenot limited to antibodies reactive to the cytokine-binding domain or anyportions thereof and which compete with the binding with cytokines.Other compounds including small molecules or synthetic or naturalchemical compounds capable of competing with the binding of a cytokineto the cytokine-binding domain or any portion thereof are also includedin the present invention.

[0089] In another aspect of the invention there is provided a method ofpreventing or treating a cytokine—related condition, said methodincluding administering to a subject an effective amount of a compound,agonist or antagonist as described above.

[0090] The compound, agonist or antagonist may be used singularly or incombination with other therapeutic agents such as corticosteroids.

[0091] The cytokine related condition may be a condition associated withany one of the group including GM-CSF, IL-3 and IL-5 or IL-4 and IL-13or a condition which requires the binding of the cytokines to a commonβ_(c) chains or to an analogous common chain in other receptors.

[0092] The examples below recite the use of antibodies to the cytokinebinding domain as antagonists. However other compounds capable ofinhibiting the binding of cytokine will be equally applicable.

[0093] The antagonist effect preferably leads to blocking of at leastone function of any one of the cytokines which may be bound to a commonβ_(c) chain or an analogous common chain. One of the benefits that isproposed to be derived from these antagonists is their use in modifyingcells stimulated by one of the cytokines, and more in one specific formmodifying the activity of the cytokines is proposed to impact greatly oncellular functions including eosinophil function. Therefore preferablythe activity leads to inhibition of stimulation of effector cellactivation and where the antibody or fragment thereof is to be used fortreatment of asthma (for example), it leads most preferably toinhibition of IL-5, IL-3 and GM-CSF or IL-4 and IL-13 mediatedeosinophil activation. It will be understood however that cells otherthan eosinophils are also the effectors of adverse conditions in humansand animals as a result of stimulation by these cytokines and inhibitionof such stimulation is also contemplated by this invention These includecells that express either one or all of GM-CSF, IL-3 and IL-5 receptors,or the IL-4 and IL-13 receptors the stimulation of which leads topathology. Examples of these are leukaemic cells, endothelial cells,breast cancer cells, prostate cancer cells, small cell lung carcinomacells, colon cancer cells, macrophages in chronic inflammation such asrheumatoid arthritis and dendritic cells for immunosuppression.

[0094] A number of different facets of eosinophil function might bemodified so that in one form IL-5, IL-3 and GM-CSF or IL-4 and IL-13mediated eosinophil survival is inhibited or blocked. In a further formIL-5, IL-3 and GM-CSF, or IL-4 and IL-13 mediated eosinophil activationis inhibited or blocked.

[0095] The treatment may be aimed at being preventative by reducing therisk of contracting the condition, or the treatment may be used toalleviate or obviate the condition. The administration of thetherapeutic agent can be any pharmaceutically acceptable form and in asuitable carrier.

[0096] It is postulated by the applicants that the construction ofcompounds that bind a portion of the B′-C′ loop of the β_(c) chainsubunit or the Tyr 421 or equivalent residue as well as the amino acidor groups of amino acids that interact with Tyr 421 or equivalentresidue in either the Domain 3 of the β_(c) chain subunit or the α chainsubunit will be therapeutically useful for intervention in conditionswhere IL-3, GM-CSF and IL-5 or IL-4 and IL-13 play a pathogenic role,mainly allergy, asthma, leukaemia, lymphoma and inflammation includingarthritis.

[0097] Similarly for other cytokine receptors it is thought thatantagonists or agonists will be therapeutically useful.

[0098] Since gp130 is functionally analogous to β_(c) in theGM-CSF/IL-3/IL-5 receptor system, in that it is a common binding subunitand signal transducer for the IL-6, oncostatin M (OSM), ciliaryneurotrophic factor (CNTF), leukaemia inhibitory factor (LIF) and IL-11,it is suggested that targeting/blocking of this cytokine binding domainwill lead to antagonism of the IL-6, LIF, OSM CNTF and IL-11. Antagonismof this receptor system will be useful in inflammation, leukaemia andlymphoma. Antagonist of IL-2Rβ/γ may be useful as immunosuppressants.Antagonists of LIFR may be useful for the prevention of implantation ofembryos in utero. Antagonist of IL-4/IL-13 will inhibit IgE productionand may be useful in treating asthma and allergies. [M. Willis-Karp etal (1998)].

[0099] Antagonist of IL-3 may be useful in treating allergy andfollicular B cell lymphoma. Antagonists of IL-4 may inhibit IgEproduction, and be useful for treatment of asthma and allergy.Antagonists of IL-6R may be useful as an anti-inflammatory and may beused to inhibit myeloma growth. Antagonists against IL-7 may be usefulas an immunosuppressant. Antagonists of the leptin receptor (OBR) may beuseful in the treatment of cachexia, weight loss in conditions such asAIDS, cancer and parasitic diseases.

[0100] Agonists agents that bind to β_(c) via the B′-C′ loop asdescribed above may be used to stimulate hemopoesis, and to boost animmune response against microorganisms and parasites. Agonist agentsthat bind to LIFR may be useful in the suppression of embryonic stemcell differentiation. Agonists agents that bind to IL-2RP may be used inimmunostimulation. Agonists agents that bind to IL-4R/IL-13 may haveanti-tumour activity.

[0101] Agonists agents that bind to specific subunits IL-3R may be usedin the in vivo and ex vivo expansion of early hemopoietic cells.Agonists agents that bind to IL-4R may have useful anti-tumour activity.Agonists agents that bind to IL-7R may have useful anti-tumour immunity.Agonists agents that bind IL-11 may prove a useful adjunct to cancertherapy. Agonists agents that bind to EPOR may be used to correctanaemia of chronic renal failure, of chronic inflammatory diseases andof malignant diseases. Agonists agents that bind to TPOR, may be usefulfor correcting thrombocytopenia (such as may be associated with chronicinflammatory diseases, malignancies, chemo- and radio-therapy).

[0102] Examples of useful agonists are those for erythropoietin andthrombopoietin to elevate erythrocyte and platelet numbers in bloodfollowing blood cell loss, chemotherapy, radiotherapy, immunosuppressionor bone marrow transplantation. Agonists of OBR may be used to induceweight loss, and in particular for obesity which is considered to be acontributing factor of hypertension, coronary heart disease andnoninsulin-dependent diabetes mellitus. The molecules whether agonist orantagonist can be isolated on the basis of their ability to interactwith the cytokine binding domain as described above.

[0103] The present invention will now be more fully described withreference to the following examples. It should be understood, however,that the description following is illustrative only and should not betaken in any way as a restriction on the generality of the inventiondescribed above.

EXAMPLES Example 1 Crystallised BION-1-D4β_(c) Complex.

[0104] D4β_(c) (residues 338-438 with an additional N-terminal Met) wasexpressed using the pEC611 vector in E. coli and purified byreverse-phase HPLC. The expressed protein was insoluble but could berecovered from the bacteria by dissolution in 6 M guanidine-HCl and 50mM sodium acetate buffer pH 4.0. After HPLC the protein was dialysedexhaustively against 5 mM MES buffer pH 6.0. The BION-1 MoAb was raisedagainst D^(4β) _(c) (Sun, Q. et al (1999) Blood, 1943) and Fab fragmentswere generated and purified by standard methods. The complex wasproduced by mixing BION-1 Fab and D4β_(βc) to give a 1:1 (mol/mol)complex which was purified on a Superdex 75 (Amersham Pharmacia) gelfiltration column. Crystals of the complex were grown by thehanging-drop vapor diffusion method at 22° C. 2 μl droplets of proteinsolution (protein concentration of 5-7 mg ml⁻¹) were mixed with 1.5 μlof the reservoir solution and equilibrated against a 1 ml reservoirconsisting of 100 mM citrate buffer pH 5.5 containing 12% (w/v)polyethylene glycol 4000. The crystals reached maximum size ofapproximately 0.6 mm×0.2 mm×0.2 mm over 10 days. The crystals belongedto space group P4₁2₁2 with cell dimensions a=b=77.6 Å, c=294.9 Å. Thecrystals were micro-manipulated, washed several times in reservoirbuffer, dissolved in SDS-Tricine sample buffer and gel electrophoresisperformed to confirm that the crystals were of the intact complex. Thecrystals proved to be sensitive to radiation and hence cryocooling wasessential. However, the crystals were fragile and an array of commonlyused cryoprotectants caused disordering of the crystals. Aflash-freezing protocol was eventually established which involvedsoaking the crystals in 5% (v/v) increments of 2-methyl-2,4-pentanediolfor two minutes till a final concentration of 15% (v/v)2-methyl-2,4-pentanediol. A native data set was collected from a singleflash-frozen crystal in-house on a MARResearch imaging plate areadetector with CuKa X-rays generated by a Rigaku RU-200 rotating anodegenerator. The cryoprotectant used was 15% MPD. The diffraction datawere processed and analysed using DENZO and SCALEPACK [Z. Otwinowski andW. Minor, (1997)] and programs in the CCP4 suite (Daresbury Laboratory,UK). The processing resulted in a 91% complete data set to 3.3 Aresolution consisting of 13,143 unique reflections. The overall R_(sym)was 12.4% and the multiplicity was 2.5. (See Crystallographic Analysisin Table 1).

[0105] The crystal structure was solved by molecular replacement usingAMoRe (Navaza, J. (1994)) and the in-house native data set.Non-redundant Fab fragments were downloaded from the protein databank(PDB) and systematically tested as molecular replacement search probes.The second search probe tested, a mouse Fab fragment with PDB identifier1YEC(Charbonnier, J. B. et a[(1997)), proved successful. The tenth peakin the rotation function (peak height of 3.3 σ) produced the highestpeak in the translation function (with a correlation coefficient of 27.9and R_(factor) of 54.1% compared with the next highest peak which had acorrelation coefficient of 17.3 and R_(factor) of 57.5%). The statisticsindicated that P4₁2₁2 was the correct enantiomorphic space group. Rigidbody refinement of the initial solution lead to a model with acorrelation coefficient of 28.7 and a R_(factor) of 49.9% (resolutionrange of 10.0 Å to 4.5 Å). Further refinement in which the Fab domainswere treated as separate rigid bodies resulted in further improvement ofthe statistics (R_(factor) of 46.1% and a drop of R_(free) from 50.9% to43.8%). Maps calculated from this solution yielded readily interpretabledensity for D4β_(c). The model of the complex was then built with thehelp of skeletonized maps using the program O (Jones, T. A. (1991)) andrefined using the maximum likelihood target in the program package CNS(Brunger, A. T. et al (1998)). The refinement was completed with thesynchrotron native data set (Table 1). In the final stages a bulksolvent correction and restrained individual isotropic B-factors wereapplied. The quality of the final map was very good with no breaks inthe main-chain connectivity and the real space fit (Jones, T. A. (1991))of residues into the map never fell below 0.7. The final model comprisesresidues 338 to 438 for D4β_(c), all residues for the Fab fragment, 124solvent molecules and one carbohydrate unit, an N-acetylglucosamineunit, off the BION-1 residue Asn^(26L) The choice of solvent moleculeswas conservative: they were only accepted if they appeared as peaks witha signal of more than 3 times the r.m.s. error in difference maps,reappeared in subsequent 2F_(o)-F_(c) maps, took part in at least onehydrogen-bonding interaction and had temperature factors less than 80Å². The stereochemical quality of the final model is good (Table 1) andother stereochemical parameters such as side-chain chi angle values,peptide bond planarity, alpha-carbon tetrahedral distortions andnon-bonded interactions are all significantly better than the allowedranges according to PROCHECK (Laskowski, R. A. et al (1993)). Thecorrectness of the tracing is supported by residue omit maps (where 10%of the model was deleted, a round of simulated annealing performed toreduce bias and the resultant map examined in the region of omission)and 3D-1D scores that never fall below 0.2 indicating no residues are inchemically unreasonable environments (Luthy, R. et al (1992)).

Example 2 Model of Complex Between α Chain, Domains 3 and 4 of β_(c) andGM-CSF, IL-5 and IL-3.

[0106] In order to understand why the β_(c) chain recognises all threecytokines, applicants have modelled the complex between α chain,domains' 3 and 4 of β_(c) and each cytokine.

[0107] The modeling proceeded as follows: (i) A structure-based sequencealignment of the cytokine-binding homology regions (CHRs) from all knownclass 1 cytokine receptor structures was performed. The amino acidsequences of the GM-CSF α chain CHR and of domain 3 of β_(c) were thenadded and manually aligned. The CHR of the α chain and domain 3 of β_(c)were built by homology to the GHR crystal structure using the multiplesequence alignment as a guide. Loop regions were constructed frompeptide fragment data bases and the models subjected to energyminimisation. The stereochemical quality of each model was judgedexcellent by the program PROCHECK [R. A. Laskowski, et al (1993)] andtheir correctness supported by 3D-1D scores that never fell below zero[R. Lüthy, et al (1992)]. (ii) The D4β_(c) structure was superimposed onthe corresponding domain of GHR. (iii) Domain 3 was connected to domain4 based on the L-shaped orientation seen in the other class 1 cytokinereceptors. The positioning was supported by the lack of steric overlapwith BION-1 which also recognises a fragment of β_(c) which includesdomain's 3 and 4, the short 2 residue linker between the domains and thefinding that the gp130 structure, which has no ligand bound, also hasthe domains in the same orientation. (iv) GM-CSF [K. Diederichs, et al(1991)] was docked onto its corresponding α chain manually using the GHRcomplex structure as a starting point and then optimising interactionsusing all the available mutagenesis data. (v) The cytokine-α chaincomplex was docked onto the modelled β chain by superimposing the GM-CSFcomponent onto the hormone of the GHR complex (using a site 2orientation in agreement with mutagenesis data. (vi) Small, rigid bodyadjustments were made manually on the cytokine-α chain complex, withrespect to D4β_(c) in order to optimise contact between cytokine andbeta chain. (vi) The other cytokines, IL-3 [Y. Feng, et al (1996)] andIL-5 μM. V. Milburn et al., Nature 363, 172 (1993)], were superimposedonto GM-CSF of the modelled complex. (vii) The hexameric complex,consisting of 2 α subunits, 2 β subunits and 2 cytokine monomers, wasconstructed from the trimeric α subunit/β subunit/cytokine model byassuming a proper twofold exists between the two trimers. (Althoughdeviations from a precise twofold relationship are quite possible, largedeviations are not envisaged since all subunits have transmembraneregions that must stay imbedded in the membrane on complex formation).The modeling, together with biochemical data yielded two solutions inwhich the second trimer was located either clockwise or anticlockwisewith respect to the first trimer when viewed down onto the membranesurface (FIG. 3). However, only one solution (FIG. 3) explained theimportance of Tyr 421. Intriguingly, each monomer of the GM-CSFcrystallographic dimer [Walter, M. R. et al (1992)] and of the IL-5covalent dimer [M. V. Milburn et al (1993)] bind to each α chain in thepreferred complex model.

[0108] The resultant model reveals the following: (i) The membraneproximal domain of the α chain homology model possesses an elongatedhydrophobic surface patch of dimensions 15 Å by 8 Å. Major contributorsto the patch are Val 224, Val 226, Cys 228, Ile 313, Phe 315 and Gly 316(GM-CSF α chain numbering). Most of these residues are conserved in αchains from different receptors and from different species (data notshown). (ii) The hydrophobic patch of the α chain is close enough to theH1 patch of D4β_(c) to envisage an interaction between the two; however,the interaction area is likely small, consistent with data showing thatthere is little or no dimer formation observed in the absence ofcytokine (Woodcock, J. M. et al (1997)). The corresponding H1 patch ofGHR is also involved in subunit contacts (DeVos, A. M. et al (1992)).There are numerous interactions between each cytokine and the B′-C′loop. Significantly, the side-chains of Tyr 365, His 367, and Ile 368form a cytokine-binding triad that converges closely at their tips toform a pivot point to which all three cytokines bind via the essentialglutamate (Glu 21 of GM-CSF, Glu 22 of IL-3 and Glu 13 of IL-5) (Hercus,T. R. et al. (1994)). These observations are consistent with mutagenesisdata that show Tyr 365, His 367 and lie 368 are key GM-CSF bindingdeterminants (Lock, P. et a. (1994)). Surprisingly, Tyr 421, the soleresidue in the F′-G′ loop implicated in high affinity binding (Woodcock,J. M. et al (1996)) is orientated away from the cytokine-binding site inthe crystal structure (FIG. 1A). Biochemical data also shows that,whilst a Tyr 421 Phe mutation has a significant effect on thephosphorylation of the cytoplasmic domain, mutations in the B′-C′ loophave a nominal effect (FIG. 2). An explanation for the critical role ofthis residue comes from the crystal structure and observations that theα,β heterodimer exists as a higher order complex (Lia, F. et al. (1996),Stomski, F. C. et al (1998)). A heterohexameric complex of two α chains,two β chains and two cytokines has been proposed based on the locationof disulfide bridges in the GM-CSF receptor complex (Stomski, F. C. etal. (1998)) and by analogy to the IL-6 receptor system which has beenshown to form a similar hexameric complex (Ward, L. D. et al. (1994)).We have modelled the hexameric complex and find that Tyr 421 is in anideal position to interact with the second α chain of the complex (FIG.3).

[0109] The monoclonal antibody antagonist, BION-1, forms extensive andintimate interactions with the receptor domain (FIG. 4 and Table 2). Thetotal surface area buried on complex formation is 1,500 Å², which is inthe range reported for other antibody-protein antigen complexes (Davies,D. R. and Cohen G. H. (1996)). In total, there are 2 salt bridges, 8hydrogen-bonding Interactions and 86 van der Waals (vdw) interactions.

[0110] The contact surface comprises 15 residues from BION-1 with 9residues from V_(H) and 6 residues from V_(L). The majority of contactsare roughly shared between four of the CDRs: CDR L1 (1 hydrogen bondsand 24 vdw contacts); CDR L3 (1 salt bridge, 3 hydrogen bonds and 20 vdwcontacts); CDR H1 (1 salt bridge, 3 hydrogen bonds and 18 vdw contacts);CDR H3 (1 hydrogen bonds and 15 vdw contacts). In addition, CDR H2provides a number of contacts (9 vdw contacts) but CDR L2 makes nocontacts with the receptor domain. Modeling suggests that CDR L2interacts with domain 3 of the intact b chain. In total, 6 residues fromthe B′-C′ loop (between residues 362 and 368) and 4 residues from theF′-G′ loop (between residues 416 and 422) are involved in antibodyinteractions. The B′-C′ loop interacts with CDRs H₁, H2, H3, L1 and L3whereas the F′-G′ loop interacts only with CDRs H3 and L1. The specificpolar interactions between the B′-C′ loop and the antibody are asfollows: Lys 362 forms a salt bridge to Asp 94L, Glu 366 forms a saltbridge to Lys 35H, and the following residues form potential hydrogenbonding interactions: Arg 364 and Tyr 33H, Arg 364 (main-chain) and Tyr33H, Tyr 365 and Glu 93L (main-chain), Tyr 365 and Asp 94L, Glu 366 andTyr 33H (main-chain), His 367 and Asn 91L (main-chain). There are twopotential hydrogen-bonding interactions involving the F′-G′: Thr 416 andTyr 28L, Arg 418 and Gly 96aH (main-chain). There is one small cavity of9.9 Å³ in the antibody-antigen interface. The cavity is lined byresidues Tyr 365, His 367 and lie 368 of the receptor and Val 27, Tyr28, Phe 32 and Asn 92 of the antibody light chain.

[0111] The B′-C′ loop of D4β_(c) is nestled in the shallowantigen-binding groove between the V_(H) and V_(L) domains whereas theF′-G′ loop forms a more peripheral interaction with CDR L1 of BION-1.Interactions from the B′-C′ loop account for 75% of the totalinteractions of D4β_(c) with BION-1. Of particular note are numerousaromatic interactions involving aromatic residues from BION-1 and Tyr365 and His 367 of the receptor (FIG. 4 and Table 2). These types ofinteractions are a common feature at antibody combining sites.

[0112] The epitope of D4β_(c) recognised by BION-1 largely overlaps thesurface that interacts with the cytokines. Furthermore, BION-1 inhibitedthe GM-CSF/IL-3/IL-5-induced proliferation of eosinophils in vitro,highlighting the feasibility of single molecule antagonists of severalcytokines. This multi-hit approach may prove useful in allergicinflammation and cancer where more than one cytokine is frequentlyassociated with these diseases. The structure presented here provides anumber of possibilities for the design of novel therapeutics: (i) Theaffinity of BION-1 for intact β chain is 50 nM in contrast to highaffinity cytokine-binding of 0.1 nM. The structure provides theopportunity of engineering a higher affinity antibody or correspondingsmall molecule mimetic, such as a cyclized, mutated version of a majorcontributing CDR from BION-1. (ii) The presence of the groove at thecytokine-binding interface is an appealing site for the design of smallmolecule antagonists. (iii) The location of Tyr 421 at a criticalsubunit interface provides another distinct target for structure-basedinhibitor design.

Example 3 Functional Roles of the B-C Loop and Tyr⁴²¹

[0113] Although the structure of D4β_(c) revealed that Tyr⁴²¹ is inclose proximity to the three residues in the B-C loop involved incytokine binding (Tyr³⁶⁵, His³⁶⁷ & Ile³⁶⁸) the side-chain is orientedaway from these possibility reflecting different functional roles.Previous experiments suggested that high-affinity binding of IL-3 wassensitive to mutation of Tyr⁴²¹ (Woodcock, J. M. (1996))but not toreplacement of individual residues in the B-C loop (Woodcock, J. M. etal (1994)). We examined whether a multiple mutation in the B-C loop ofthe residues implicated in binding GM-CSF and IL-5 would affect IL-3high-affinity binding. The results showed (FIG. 6A) that alaninesubstitution of residues 365 to 368 in the B-C loop abrogatedhigh-affinity binding of both GM-CSF and IL-3. We next examinedphosphorylation of cytoplasmic tyrosine residues as this is a verysensitive measure of recruitment of β_(c) to a ligand/α-chain complex.Analogs of β_(c) that are unable to affinity-convert due to the affinityof the α/β complex for cytokine being less than or equal to that ofα-chain alone may nevertheless exhibit differences in tyrosinephosphorylation. We examined the effects of mutating the B-C loop orTyr⁴²¹, either separately or in combination, on the ability of β_(c) toundergo tyrosine phosphorylation in response to IL-3. We found thatsubstitution of Tyr⁴²¹ had a pronounced effect with high levels oftyrosine phosphorylation of β_(c) being achieved only at 3 μM IL-3, aconcentration about 500-fold higher than required by the native receptor(6 nM). In contrast, mutation of the B-C loop alone did not impairIL-3-induced phosphorylation of β_(c) at the high concentration used.Nevertheless, a role for the B-C loop in β_(c) activation wasdemonstrated by a combined mutant of the B-C loop and Tyr⁴²¹ (FIG. 6B)which abrogated IL-3-induced tyrosine phosphorylation of β_(c).

Example 4 Antagonist Interactions with the β_(c) Activation Domain

[0114] A detailed analysis of the structure of the BION-1/D4β_(c)complex confirmed and extended these observations with BION-1 seen toform extensive and intimate interactions with the receptor activationdomain (FIGS. 1A,B and 7). The total surface area buried on complexformation is 1,500 Å², which is in the range reported for otherantibody-protein antigen complexes(Davies, D. R. and Cohen, G. H.(1996)). In total, there are 2 salt bridges (Lys³⁶²/Asp^(L94) andGlu³⁶⁶/Lys^(H35)), 8 potential hydrogen-bonds and 124 van der Waals(vdw) interactions (Table 2). The B-C loop of D^(4β) _(c) is nestled inthe shallow antigen-binding groove between the V_(H) and V_(L) domainswhereas the F-G loop forms a more peripheral interaction with CDR L1 ofBION-1 (FIGS. 1A,B and 7). The contact surface comprises 14 residuesfrom BION-1 with 9 residues from V_(H) and 5 residues from V_(L). Themajority of contacts are roughly shared between four of the CDRs: CDR L1(1 hydrogen bond and 29 vdw contacts); CDR L3 (1 salt bridge, 3 hydrogenbonds and 28 vdw contacts); CDR H1 (1 salt bridge, 3 hydrogen bonds and36 vdw contacts); CDR H3 (1 hydrogen bond and 23 vdw contacts). Inaddition, CDR H2 provides a number of contacts (8 vdw contacts) but CDRL2 makes no contacts with the receptor domain. In total, 6 residues fromthe B-C loop (between residues 362 and 368) and 3 residues from the F-Gloop (between residues 416 and 422) of D^(4β) _(c) are involved inantibody interactions with those from the B-C loop accounting for 75% ofthe total. The B-C loop interacts with CDRs H1, H2, H3, L1 and L3whereas the F-G loop interacts only with CDRs H3 and L1 (FIG. 4). Thereis one small cavity of 9.9 Å³ in the antibody-antigen interface. Thecavity is lined by residues Tyr³⁶⁵, His³⁶⁷ and Ile³⁶⁸ of the receptorand Val²⁷, Tyr²⁸, Phe³² and Asn⁹² of the antibody light chain. Not allof the potential salt bridges and hydrogen bonds identified above arelikely to contribute productively to complex formation sincesubstitution analysis has only identified Glu³⁶⁶, Arg⁴¹⁸ and Met³⁶³ orArg³⁶⁴ in D4β_(c) as contributing to the epitope for binding BION-1(Sun, Q. et al (1999) Blood, 1943) (Table 2).

[0115] Finally it is to be understood that various other modificationsand/or alterations may be made without departing from the spirit of thepresent invention as outlined herein.

REFERENCES

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What is claimed:
 1. A cytokine-binding domain or portion thereof whichbinds to at least one cytokine and is capable of transducing a cytokinesignal through a single cytokine receptor, said domain comprising aportion of the B′-C′ loop of Domain 4 of β_(c) chain or analogousstructure of a cytokine receptor.
 2. A cytokine binding domain accordingto claim 1 comprising a portion of the B′-C′ loop of domain 4 and agroove which is defined by the B′-C′, F′-G′ loops and the N-terminalsection of Domain
 4. 3. A cytokine binding domain according to claim 1further including a Tyrosine residue capable of interaction with an αchain subunit or with Domain 3 of the β_(c) chain subunit to allow highaffinity binding of the cytokine.
 4. A cytokine binding domain accordingto claim 3 wherein the tyrosine is Tyr42¹ or equivalent residue of ananalogous common signalling structure.
 5. A cytokine binding domainaccording to claim 1 wherein the B′-C′ loop of Domain 4 comprisesresidues 365 to 368 forming a type 1 β-turn or an analogous structure inan analogous common signalling structure.
 6. A cytokine binding domainaccording to claim 1 wherein the binding domain of β_(c) or portionthereof which binds to at least one cytokine is defined by an areabordered by any one of the following residues including Lys362, Tyr365,His367, Ile368, Arg418, Gly420, Asn422, Thr416, Ile338, Gln339, Met340and Met361 or equivalent residues in an analogous common signallingstructure of a cytokine receptor.
 7. A cytokine binding domain accordingto claim 1 wherein the B′-C′ loop of the Domain 4 includes Tyr365,Ile368 and His367.
 8. A cytokine binding domain according to claim 1that binds to at least two cytokines selected from the group includingIL-3, IL-5 and GM-CSF, or IL-4 and IL-13.
 9. A cytokine binding domainaccording to claim 1 wherein the common β_(c) chain or analogousstructure of a cytokine receptor is derived from any one of thefollowing, including GM-CSF, IL-3 and IL-5 receptors, the common IL-2receptor γ chain (shared by the IL-2, IL-4, IL-7, IL-9 and IL-15receptors) and gp130 (shared by the IL-6, IL-11, LIF, ciliary neutrophicfactor, oncostatin M and cardiotrophin receptors) or from any of thecytokine superfamily receptors but not limited to the group comprisingLIFR, gpl30, IL-2Rβ, IL-4R/IL-13R, IL-2Rγ, IL-3Rα, EPOR, TPOR and OBR oris selected from a related (class 1) cytokine receptor structureselected from the group including but not limited to growth hormonereceptor (GHR), prolactin receptor (PRLR), erythropoietin receptor(EPOR), G-CSF receptor (G-CSFR) and gp130.
 10. A cytokine binding domainaccording to claim 9 wherein the common 62 _(c) chain is derived fromthe IL-5, IL-3 or GM-CSF receptor.
 11. A cytokine binding domainaccording to claim 2 wherein the F′-G′ loop adopts a type IVβ turn atits tip in Domain 4 and includes the residues Arg418 and Tyr421.
 12. Amethod of identifying a compound having cytokine agonist or antagonistactivity which comprises: subjecting a potential cytokine agonist and/orcytokine antagonist compound to a cytokine binding domain or portionthereof according to claim 1; and determining the presence of an agonistor antagonist response to the compound on the activity of a cytokine.13. A method of identifying a compound having a cytokine antagonistactivity, which comprises: subjecting a potential cytokine antagonist toa cytokine binding domain or portion thereof according to claim 1; andidentifying a compound that has bound to the cytokine-binding domainwherein said compound has an antagonist response on the activity of thecytokine.
 14. A method according to claim 12 or 13 wherein the cytokineis selected from the group including IL-3, IL-5 and GM-CSF; or IL-4 andIL-13 and the presence of an agonist or antagonist is determined by theability of the agonist or antagonist to activate or inhibit an IL-3,IL-5 or GM-CSF, IL-4, IL-13 response.
 15. A method according to claim 12or 13 wherein the cytokine agonist or antagonist further binds to Tyr421or an equivalent residue of a common signalling unit of a cytokinereceptor.
 16. A cytokine agonist or antagonist identified by a methodaccording to claim 12 or
 13. 17. An antibody or fragment thereof to acytokine binding domain according to claim
 1. 18. A cytokine bindingdomain according to claim 1 comprising a mutation directed to any one ofthe residues selected from the group including Gln340, Ile338 and Met361or an equivalent residue of a common signalling unit of a cytokinereceptor.
 19. A method of preventing or treating a cytokine-relatedcondition, which method comprises administering to a subject aneffective amount of an agonist or antagonist according to claim
 16. 20.A method of preventing or treating a cytokine-related condition, whichmethod comprises administering to a subject an effective amount of anantibody according to claim
 17. 21. A method according to claim 19wherein the cytokine-related condition is selected from the groupincluding survival or activation of eosinophil function, asthma,leukemia, breast cancer, prostate cancer, small cell lung carcinoma,colon cancer, chronic inflammation including rheumatoid arthritis,immunosuppression, allergy, lymphoma, and cachexia., wherein saidcytokine agonist or antagonist is an antagonist.
 22. A method accordingto claim 20 wherein the cytokine-related condition is selected from thegroup including survival or activation of eosinophil function, asthma,leukemia, breast cancer, prostate cancer, small cell lung carcinoma,colon cancer, chronic inflammation including rheumatoid arthritis,immunosuppression, allergy, lymphoma, and cachexia.
 23. A methodaccording to claim 19 wherein the cytokine-related condition is allergicinflammation and the antagonist inhibits the binding of any one of IL-5,IL-3 or GM-CSF to the IL-5, IL-3 or GM-CSF receptor.
 24. A methodaccording to claim 20 wherein the cytokine-related condition is allergicinflammation and the antagonist inhibits the binding of any one of IL-5,IL-3 or GM-CSF to the IL-5, IL-3 or GM-CSF receptor.
 25. A methodaccording to claim 23 wherein the allergic inflammation results inasthma.
 26. A method according to claim 24 wherein the allergicinflammation results in asthma.
 27. A method according to claim 19wherein the cytokine-related condition is selected from the groupincluding hemopoesis, boosting immune response, suppression of embryonicstem cell differentiation, immunostimulation, antitumor activity,expansion of early hemopoietic cells, anemia, correctingthrombocytopenia, wherein said cytokine agonist or antagonist is anagonist.